Tuberculosis (TB) is an infectious disease that is caused by Mycobacterium tuberculosis (Mtb) (1). The disease is spread through the air when infected individuals expel bacteria (1). Despite the fact that recent efforts to eradicate TB have had positive results, the global burden of the disease remains very high (1, 2), with 9 million new cases of tuberculosis and 1.4 million deaths worldwide in 2011. The persistence of Mtb in the host requires a persister state which effectively shuts down many biological processes that are targets for common antibiotics (3-5). This persister state can be combatted by the inclusion of pyrazinamide (PZA) in the drug regimen (3, 6) and this drug is responsible for shortening the duration of the chemotherapy from 9-12 months to 6 months, due to its unique selectivity for the persister state. Despite the long term use of PZA as a first line TB drug, its molecular target remains unclear. Recently, a new target for PZA has been identified as ribosomal protein S1, or RpsA (7). RpsA is a vital ribosomal protein that plays a significant role in messenger RNA (mRNA) translation (8, 9) and in trans-translation, a unique process that uses a transfer-messenger RNA (tmRNA) molecule to rescue stalled ribosomes (10, 11). In Mtb RpsA contains four consecutive S1 RNA- binding domains and a C-terminal domain (CTD) that is unique to actinobacteria. Recent findings have shown that RpsA overexpression confers PZA resistance in Mtb and select PZA-resistant Mtb clinical isolates harbor mutations that map to the rpsA gene. Additionally, clinical isolates of a pathogenic bacterium belonging to Mtb complex, M. canettii (Mca), are intrinsically resistant to PZA and contain mutations in the rpsA gene (12). Previous biochemical work confirmed that the active form of PZA, pyrazinoic acid (POA), binds to wild-type RpsA and inhibits trans-translation, however, a clinically-relevant mutation in the CTD abrogates POA binding (7). It is not clear how POA binds to RpsA and inhibits trans-translation. We hypothesize that POA binding to RpsA interferes with tmRNA binding resulting in an inhibition of trans-translation, a process that is highly important for the persister state o Mtb. In order to test this hypothesis and elucidate the mechanism of POA activity, we will test tmRNA-mediated peptide tagging and the drug susceptibility of this process. Additionally, we will biochemically and biophysically characterize the mutant RpsA proteins that are found in drug resistant clinical isolates. Furthermore, we will structurally characterize RpsA in order to gain insight into the molecular determinants of POA binding and POA resistance mutations. These studies will elucidate a mechanism of action for the unique persister drug PZA and have implications for the design of much needed and more powerful persister drugs for improved treatment of TB and drug-resistant TB.